Endo-β-D-xylanase (EC3.2.1.8) activity was determined using the modified dinitrosalicylic acid (DNS) method (Bailey et al., 1992). Briefly, 100 μL of the culture supernatant solution and 900 μL of a 1% (w/v) beechwood xylan (Sigma-Aldrich Pvt. Ltd., United States) suspension in 50 mM citrate buffer (pH 4.0) were mixed. The incubation of mixture was carried out at 55°C for 5 min and with the addition of 1000 μL DNS, the reaction can be stopped. The xylanase activity unit is the needed amount of enzyme to release 1 μmol xylose equivalent in every 60 s.

Protein concentrations were determined using Lowry’s method (Lowry et al., 1951), and BSA (bovine serum albumin) used as the standard was purchased from Roche (738328).

Each purification process was executed at 4°C. The crude enzyme was first purified with 20–50% ammonium sulfate (Sinopharm Chemical Reagent CO., Ltd.); the preliminary purified enzyme solution was subjected to dialysis for 12 h with 20 mM sodium acetate buffer, pH 3.8, and then loaded on a SP-Sepharose Fast Flow ion-exchange column equilibrated with 20 mM sodium acetate buffer, pH 3.8. The elution was carried out with a linear NaCl gradient (0–100 mm in 20 mM sodium acetate buffer, pH 3.8) at a flow rate of 1.0 ml/min.

All active fractions were concentrated to 1.0 ml and slowly loaded on a Q-Sepharose Fast Flow column, which was equilibrated with 20 mM citrate buffer (pH 5.6). At a flow rate of 1.0 ml/min, 2 ml fractions were collected after eluting protein. Fractions exhibiting xylanase activity were then collected for further use.

Enzyme purity and molecular weight can be determined by SDS-PAGE and 12.5% (w/v) separation and 4.5% (w/v) stacked gel (Laemmli, 1970). By Coomassie Brilliant Blue G 250, the gel was stained. And the molecular weight of the enzyme is determined with a low molecular weight scale label.

The xylanase activity was detected by incubation with a zymogram gel containing 1% (w/v) birchwood xylan (Sigma-Aldrich Pvt. Ltd., United States) at 40°C for 2 h, followed by staining in Congo red solution (1 mg/ml). The dye was removed using 1 M NaCl solution till clear areas in a dark red background appeared, which is indicative of xylanase activity (Schwarz et al., 1987).

With 1% beechwood xylan as the substrate, the best pH value required in xylanase activity can be determined at 55°C in the pH range of 2.70–9.00. The highest xylanase activity was used to define 100% activity. The following buffer systems (50 mM) were used: citrate buffer, pH 2.70–5.70; acetate buffer, pH 3.20–5.80; MES buffer, pH 5.20–7.20; MOPS buffer, pH 6.20–8.20; Tris-HCl buffer, pH 7.00–9.00.

The effect of pH on the stability of xylanase was evaluated by incubating the enzyme at different pH values for 30 min at 50°C. Samples were then cooled with ice water. Afterwards, residual activity of each sample was determined under standard conditions.

The optimal temperature required for enzyme activity was determined in the range of 40–60°C. In each case, samples were diluted in the optimal pH buffer solution prior to xylanase activity assay. The thermal stability of the immobilized and free xylanase were determined by incubating the enzyme in the optimal pH buffer solution for 5 h at different temperatures (40–60°C). At certain time intervals, aliquots could be extracted, and under standard conditions, residual activity can be tested. The 100% activity can be determined by the unheated enzyme.

Ultrasonic hydrolysis was carried out using a DTD5200S ultrasonic device (Beijing Hongxianglong Biotechnology Co., Ltd.) with an ultrasonic power of up to 300 W, a frequency of 135 kHz, and a microtip diameter of 10 mm. The instrument used was a US bath type reactor with five different 2.0 cm ultrasound generator probes at four different frequencies (28, 40, 50, and 128 kHz); all probes can deliver maximum power of 300 W. Prior to hydrolysis, XAC was powdered into 0.45 mm particles. Subsequently, the ground XAC were dispersed at liquid to solid ratios of 50:1 (mL:g), in 10 ml 50 mM acetate buffer (pH 3.7) and the reaction was performed using 16U/mL acidic xylanase at 40°C for 40min. The prepared XAC was completely mixed in a beaker 3 cm shorter than the water bath, and the probe of the ultrasonic generator (about 1 cm to the liquid level) was inserted into the ultrasonic field immediately. The ultrasound intensity released from the probe was regulated to 0.04, 0.13, 0.22, 0.31, 0.40 W/cm² and the ultrasound frequencies was regulated to 28, 40, 50, and 128 kHz. Each treatment was replicated thrice.

The function of ultrasound (0.31 W/cm² and 50 kHz) on acidic xylanase was evaluated. The enzyme activity of xylanase at 40°C for 5 min was determined by different substrate concentrations, and the kinetic parameters of xylanase on beech xylan were determined; with the help of “GraFit” software, we calculated Km and Vmax values.

Enzyme XynB (0.20 mg/ml) in 0.05 M acetate buffer (pH 3.8) was sonicated at 40°C using three categories or protocols for monitoring changes in enzyme structure under different ultrasound conditions. The first group of experiments studied the effect of ultrasound with different sound intensity (0.04, 0.13, 0.22, 0.31, 0.40 W/cm²) at 50 kHz for 40 min. The second set of experiments was performed at different ultrasonic frequency (28, 40, 50, 128 kHz) at 0.31 W/cm² for 40min. The third set of experiments was performed at different time points (10, 20, 30, and 40 min) at 0.31 W/cm² and 50 kHz.

After ultrasound, CD spectra were recorded by spectrometer (JASCO, Tokyo, Japan; J-815 type), and CD spectra were recorded by a quartz container with 1 mm optical path length at room temperature (20 ± 1°C). CD spectra were scanned in the far ultraviolet (260–190 nm) range with a repetition rate of 100 nm/min and a bandwidth of 0.1 nm. CD data are represented by molar ellipticity in mde. cm². dmol⁻¹. The α-helix in the measured molar ellipticity of xylanase was observed at 208 nm (Wang et al., 2003).

The particle sizes of xylan (XAC) and its aggregates in XAC suspension and acoustic XAC suspension were measured by TopSizer laser diffraction particle size analyzer (OMEC, Zhuhai, China). Which can provide useful information for comparing the size of non-spherical rod like xylan whiskers and their aggregates before and after sonication.

The morphology of XAC particles as well as ultrasonic pretreated samples was observed with a VEGA\\LSU scanning electron microscope (Tescan Company, Czech Republic) at 15 kV accelerating voltage. The samples were covered with a thin layer of gold as a conductive medium under a scanning electron microscope.

By nonlinearly fitting the kinetic data to the Michaelis-Menten equation, the kinetic parameters, maximum reaction rate (V _max), Michaelis constant (K _M ), and catalytic efficiency (V _max /K _M ) can be calculated. The V _max, K _M , and V _max/K _M of XynB were 189.20 ± 6.15 mg ml⁻¹ min⁻¹, 23.12 ± 0.98 mg ml⁻¹, and 8.18 ± 0.35 min⁻¹, respectively. Sonication increased the V _max and V _max /K _M by 41.1 and 23.5%, respectively. The combination of XynB and ultrasound improved the catalytic ability. V _max shows the extreme rate of the enzyme reaction when the substrate is saturated, while the K _M value shows the enzyme affinity for substrate. The increase in V _max indicated strengthening of the xylan-xylanase complex interaction, and accelerated hydrolysis, whereas a slight decrease in K _M indicated an enhancement in the affinity between XynB and XAC, possibly because of better exposure of the xylanase active site after ultrasonication (Dalagnol et al., 2017). That is, ultrasound can increase the rate of reaction and enzyme substrate affinity, and enhance the reaction by enhancing the binding of the enzyme to the substrate. It is produced by mechanical effects of ultrasonic cavitation, and ultrasound’s capability in increasing the mass transfer rate in enzymatic reactions has been reported to be supported (Sajjadi et al., 2017).

Most enzymes are monomeric spherical proteins whose catalytic activity depends on their natural configuration. Ultrasound may alter the secondary structure of the enzyme, thereby exposing the active site of the enzyme to a better extent (Subhedar and Gogate, 2014). The changes of secondary structure of proteins can be analyzed by CD spectroscopy, which calculates the α-helix, β-strand (β-Sheet and β-turn) and random coiling of xylanase, and the relationship between enzyme activity and secondary structure is determined (Li et al., 2015). A typical characteristic of all natural enzyme structure cd spectra is that the XynB spectrum has a fixed minimum peak at 209 nm and a positive peak at 195 nm, which is essentially the same as the native zymogram seen from early researches (You et al., 2010). The characteristics of proteins rich in β-chain (β-Sheet and β-turn) secondary structures can be found in these two spectra. The comparison of secondary structure content obtained using deconvolution of CD revealed an obvious difference regarding β-sheet, β-turn, and random coil. This analysis showed a slight decrease in XynB β-strand content and increase in random coil content with increase in ultrasonic intensity (from 0.04 W/cm² to 0.40 W/cm²), which was believed to be related to acceleration of enzymatic hydrolysis. Similar results were obtained when ultrasonic frequency and treatment time were altered (Data was not shown). These results indicated that ultrasonic pretreatment cast no effects on natural secondary structure of XynB, while destroying the hydrophobic interaction between protein molecules, making more internal hydrophobic groups and regions exposed to external environment.

The reduction in size is due to the high shear forces caused by ultrasonic cavitation in liquid mediums (Trujillo and Knoerzer, 2011). After dialysis, the same volume of XAC suspension was sonicated at different times (0–40 min) and then characterized by laser diffraction. A comparison of the particle size distribution of the ultrasonic suspension (Figure 4) shows that the longer the ultrasonic action time, the lower the peak position of the volume distribution (76.43 μm). When the nanoparticles appeared at 40 min (from 84.79 to 65.04 μm), they expanded to smaller particles (Alvira et al., 2010).

The microstructure of XAC prior to ultrasound pretreatment was observed using scanning electron microscopy (SEM) as shown in Figure 5. After ultrasonic pretreatment, the XAC structure was loosened and more disordered structures and irregular fragments appeared, which changed the original (0 min) unvoiced XAC suspension with large area and high density. In particular, after long-term ultrasonic pretreatment (40 min), cracks appeared on the surface of XAC; microparticles and small fragments increased the specific surface area of XAC molecules, increasing the probability of contact between the substrate and the enzyme, thereby enhancing the hydrolysis of the enzyme.

Xylan exists in one form and can be linked to other xylan structures and lignin by linking the arabinose-based ferulic acid bridge on the xylan backbone, while the lignin-saccharide complex in the corncob mostly composed of lignin-xylan complex (Van et al., 2011; Ju et al., 2013). Based on the SEM micrographs obtained in this study and calculation of the yield of XOS, we believe that sonication mainly resulted in fiber disruption, which eased subsequent enzymatic hydrolysis. This was in agreement with the observations of Jiang et al. who compared the effects of ultrasound on the functional and structural properties of black-bean protein isolate dispersions (Jiang et al., 2014).